Late coronary BVS malapposition and aneurysm: A Time for Appraisal

Late strut malapposition of currently available stents is a major issue, associated with an increased risk of stent thrombosis. To this day, biovascular scaffolds showed an increased immediate risk of struts malapposition, that however resulted reduced after a few months. Little is known about late acquired struts malapposition of biovascular scaffolds. We here describe two cases that recently occurred in our patients, and we speculate on possible mechanisms for this unexpected and potentially lethal complication. © 2015 Wiley Periodicals, Inc.     

Fracture with the newer bioresorbable vascular scaffolds

Bioresorbable vascular scaffolds (BVS) are being increasingly used in complex, real‐world lesions. The possibility of strut fractures is a rare entity with the use of drug eluting stents (DES), but has not yet been adequately described for the BVS technology. In this report, we present a case of scaffold fracture of DEsolve BVS (Elixir Medical Corporation, USA) that was diagnosed both angiographically and by optical coherence tomography(OCT). Scaffold fracture with the newer BVS is a new Achilles' heel in the BVS technology. This gap in knowledge about the etiology such as over‐expansion and potential predictors of strut fracture need to be deeply investigated before a widespread use of such technologies is adopted. © 2017 Wiley Periodicals, Inc.     

Anatomical features and management of bioresorbable vascular scaffolds failure: A case series from the GHOST registry

The Absorb bioresorbable vascular scaffold (Absorb BVS, Abbott Vascular, Santa Clara, California) promises to address some of the residual shortcomings of existing metallic stents, such as late events induced by permanent caging of the coronary vessel. Scaffold restenosis (ScR) of BVS has been poorly described so far and treatment strategies for this event remain to be codified. We report on a case series of 14 lesions in 12 patients presenting with ScR and discuss their anatomical features and management strategies. © 2015 Wiley Periodicals, Inc.     

Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo

Here, we report the development of target-specific binding proteins based on the kringle domain (KD) (∼80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-α (TNFα) and efficiently neutralizes TNFα-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.     

Impact of the bioresorbable vascular scaffold surface area on on-treatment platelet reactivity

While promising data with the novel bioresorbable vascular scaffold (BVS) are accumulating, signals of scaffold thrombosis (ST) were noted in recent reports. We aimed to assess the relationship between the total surface area (TSA) of implanted everolimus-eluting BVSs and the on-treatment adenosine diphosphate (ADP)-induced platelet reactivity in patients undergoing percutaneous coronary intervention (PCI). 202 consecutive patients undergoing BVS implantation and platelet function testing were included. For investigating the impact of the scaffold surface on platelet reactivity, patients were stratified into two groups regarding the median BVS TSA. The on-treatment ADP-induced platelet reactivity was determined with the Multiplate analyzer and 30-day follow-up was available in 98% of patients. ADP-induced platelet aggregation values (median, [IQR]) did not differ between the two study groups (12.0 [9.0–19.0] U for patients with TSA > 1.39 cm² and 13.0 [9.0–19.5] U for patients with TSA ≤ 1.39 cm²; p = 0.69). No correlation was observed between the BVS TSA and levels of platelet reactivity (Spearman rank correlation = ?0.10, p = 0.16). At 30 days after PCI, two early STs (1%) were documented. Thus, in patients on a dual antiplatelet treatment regimen following BVS implantation, the extent of blood-to-BVS contact surface does not negatively affect levels of on-treatment platelet reactivity.     

Calcineurin increases glucose activation of ERK1/2 by reversing negative feedback

In pancreatic β cells, ERK1 and ERK2 participate in nutrient sensing, and their activities rise and fall as a function of glucose concentration over the physiologic range. Glucose metabolism triggers calcium influx and release of calcium from intracellular stores to activate ERK1/2. Calcium influx also activates the calcium-dependent phosphatase calcineurin, which is required for maximal ERK1/2 activation by glucose. Calcineurin controls insulin gene expression by ERK1/2-dependent and -independent mechanisms. Here, we show that, in β cells, glucose activates the ERK1/2 cascade primarily through B-Raf. Glucose activation of B-Raf, like that of ERK1/2, is calcineurin-sensitive. Calcineurin binds to B-Raf in both unstimulated and stimulated cells. We show that B-Raf is a calcineurin substrate; among calcineurin target residues on B-Raf is T401, a site of negative feedback phosphorylation by ERK1/2. Blocking calcineurin activity in β cells prevents dephosphorylation of B-Raf T401 and decreases B-Raf and ERK1/2 activities. We conclude that the major calcineurin-dependent event in glucose sensing by ERK1/2 is the activation of B-Raf.     

Coronary artery aneurysm after implantation of a bioresorbable vascular scaffold: Case report and literature review

A 55‐year‐old man underwent successful percutaneous coronary intervention for the middle left circumflex artery with a 3.5 × 28‐mm bioresorbable vascular scaffold (BVS). At 18 months, follow‐up coronary angiography showed ectatic change with aneurysm formation over the BVS. Optical coherence tomography revealed absence of strut continuity at the aneurysm site, in the middle of the BVS. A literature review identified nine patients with intrascaffold aneurysm, including the present patient, which developed 6–32 months after BVS implantation. Of these nine patients, four underwent percutaneous coronary intervention for chronic total occlusion. The pathogenesis of coronary artery aneurysm is multifactorial. Most patients receive no further intervention, but long‐term dual antiplatelet therapy is sometimes prescribed in conjunction with regular follow‐up. © 2017 Wiley Periodicals, Inc.     

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1^EK) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1^null) mouse. In contrast to Exo1^null/null mice, Exo1^EK/EK mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1^null mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.Exonuclease 1 (EXO1) belongs to the XPG/Rad2 family of metallonucleases and was first described as a 5′–3′ exonuclease associated with meiosis in Schizosaccharomyces pombe (1). Since then EXO1 has been implicated in a multitude of eukaryotic DNA metabolic pathways and in maintaining genomic integrity. It is involved in DNA mismatch repair (MMR) by hydrolyzing DNA mismatches (2–4), in DNA double-strand break repair (DSBR) through DNA end resection (5–7), in B-cell development through the generation of antibody diversity (8), and in telomere maintenance by promotion of telomeric recombination (9). Biochemical analysis had shown that the N-terminal half of EXO1 possesses 5′–3′ exonuclease and 5′ flap-endonuclease activities (10). However, these apparently distinct functions now are thought to be mechanistically unified (11).MMR is essential for maintaining the integrity of eukaryotic genomes by removing misincorporated nucleotides that result from erroneous replication. During MMR, the repair of distinct types of mismatches is initiated by two partially redundant MutS homolog (MSH) complexes: the MSH2–MSH6 (MutSα) heterodimer, that recognizes and binds to single-base mispairs and single-base insertion/deletions, and the MSH2–MSH3 (MutSβ) complex that primarily interacts with single-base and larger insertions/deletions. Subsequent to mismatch recognition by the MSH complexes, a MutL homolog (MLH) complex consisting of MLH1-PMS2 (MutLα) is recruited to activate subsequent repair events in an ATP-dependent manner (12–14). In addition, genetic studies indicate that a second MutL complex consisting of MLH1–MLH3 (MutLγ) plays a role in the repair of a proportion of insertion/deletion mutations (15, 16). EXO1 interacts with MutSα and MutLα both in yeast and humans (2, 17). Biochemical analysis attributed a role for EXO1 in the 5′- and 3′-directed excision of the nascent mismatch-containing DNA strand downstream of MMR protein recruitment (3, 4). It has been assumed that excision by EXO1 is dependent on its nuclease activity despite the lack of clear in vivo evidence. On the other hand, studies in yeast suggested a nuclease-independent function for EXO1 as an adapter or structural scaffold in the formation of MMR protein complexes (18–20).MMR proteins facilitate the immune response because they participate in an error-prone process that promotes the affinity maturation of antibodies by increasing somatic hypermutation (SHM) at Activation-induced deaminase (AID)–induced U:G mismatches at the Ig locus (21). Conversely, defects in MMR can lead to increased mutation rates elsewhere in the genome and are associated with hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) and 15–25% of sporadic colorectal cancers (CRCs) in humans (22–24). Because of the involvement of EXO1 in MMR, it was speculated that EXO1 mutations might contribute to HNPCC or CRC. However, the role of EXO1 in suppressing CRC remains unclear despite EXO1 germ-line mutations being found in patients with atypical HNPCC (25, 26).Like DNA mismatches, double-strand breaks (DSBs) are a form of genotoxic lesions. An early response to DSBs is 5′–3′ DNA end resection, which generates ssDNA that evokes the checkpoint and homologous recombination (HR) responses. Although the identity of DNA helicases and nucleases that process DSBs are not yet as well defined in humans as in yeast (5, 6), studies from both species suggest a two-step model for DNA DSB processing. MRE11-RAD50-NBS1 (MRN) and CtIP initiate the end-trimming of the DSB, which is followed by the generation of longer stretches of ssDNA by either EXO1 or the Bloom syndrome protein (BLM)–DNA2 helicase–nuclease complex (5, 27). Deficiencies in DSBR lead to chromosomal instability, infertility, neurodegeneration, tumorigenesis, premature aging, and a decrease in class switching in the immune system that requires nonhomologous end joining (NHEJ) (28). However, the way in which EXO1 is involved in all these processes remains unclear.Previous yeast studies suggest both exonuclease-dependent and -independent functions for EXO1 in MMR and meiosis (18, 29), but the implications of distinct EXO1 functions in these biological processes remain ambiguous. We therefore generated and analyzed two mouse lines to assess the role of the structural and enzymatic functions in vivo. One line carries a HNPCC-modeled E109K knockin mutation in the exonuclease domain of EXO1 (termed Exo1^EK). The other line carries an Exo1-null knockout mutation leading to the complete loss of EXO1 protein expression (termed Exo1^null).     

Receptor for activated C-kinase 1 regulates the cellular localization and function of ABCB4

Aim:  Multidrug resistance protein 3 (MDR3/ABCB4), located on the bile canalicular membrane of hepatocytes, is responsible for the translocation of phosphatidylcholine across the plasma membrane, and its hereditary defect causes liver disorders, such as progressive familial intrahepatic cholestasis type 3. We aimed to identify the proteins responsible for the surface expression of human ABCB4 .
Methods:  We performed yeast two-hybrid screening with the cytoplasmic linker region of ABCB4 against a human liver cDNA library. This screening allowed us to identify the receptor for activated C-kinase 1 ( RACK1 ) as a novel binding partner of ABCB4 . The association of RACK1 with the linker region of ABCB4 was further confirmed by GST-pulldown assay, although we could not find out the interaction of full length of ABCB4 and RACK1 in co-immunoprecipitation assay in HeLa cells.
Results:  Down-regulation of endogenous RACK1 expression by siRNA in HeLa cells resulted in the localization of ABCB4 in the cytosolic compartment as well as reduced protein expression of ABCB4 , although mRNA expression and the protein stability of ABCB4 were not affected by the suppression of endogenous RACK1 . Similar alterations in cellular localization of ABCB4 were also found by suppressing endogenous RACK1 expression in HepG2 cells. Consequently, ABCB4 -mediated phosphatidylcholine translocation activity was significantly reduced when endogenous RACK1 expression was suppressed in HeLa cells. In contrast, the membrane surface localization and the protein expression of ABCB1 were not affected by the suppression of endogenous RACK1 expression.
Conclusion:  These results suggest that RACK1 may have a functional significance as a regulatory cofactor of ABCB4 and is indispensable for the plasma membrane localization and translocation function of ABCB4 .